Prior to this study, we downloaded the GEO database from Pubmed for analysis of the relation between AZGP1 and metastasis in sarcomas. Involving 105 STS microarray analysis, GDS2736 indicated that AZGP1 expression in sarcomas with high metastatic potential was significantly lower than that in lipoma and well-differentiated liposarcoma (WDLPS) with no or rare metastasis (Fig. 1a).
Patients and tissue specimens
Tumor samples from 86 patients with primary STS, who underwent surgery between 2007 and 2014, were obtained from the tissue bank of our institution and snap-frozen in liquid nitrogen immediately after surgical resection until RNA extraction. Paraffin-embedded specimens were used for IHC staining. To be included in our study, cases should be histologically diagnosed as grade 2 or 3 according to the FNCLCC grading system, with no preoperative chemotherapy. Metastasis, overall (OS), 4-year, metastasis-free (MFS), as well as disease-specific (DSS) survivals, were monitored with a mean follow-up of 45 months (range 23–83 months). Sample acquisition was approved by the Ethics Committee of the Hospital. Written informed consent was obtained from all patients.
RNA extraction and quantitative RT-PCR
Quantitative RT-PCR was used to detect the expression levels of AZGP1 mRNA in 81 cases of tumor tissues, since 5 cases were excluded due to RNA extraction failure. Total RNA was extracted from frozen tissues containing > 80% STS cells by RNA Extraction Kit (QIAgen) according to the manufacturer’s instructions. Quantity and quality of RNA was confirmed by a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). RNA purity was determined by an OD260/280 value between 1.8 and 2.0. For mRNA expression, cDNA was obtained from 2 μg total RNA using Moloney murine leukemia virus reverse transcriptase (M-MLV RT) (Invitrogen, Carlsbad, CA) with oligodT15 primers. GAPDH mRNA was used as an endogenous control to normalize for AZGP1 mRNA expression. qRT-PCR was performed using SYBR® Green PCR Master Mix (TOYOBO) on the ABI 7500 Fast (Applied Biosystems). Data were calculated as relative quantification to GAPDH, based on calculations of 2−△Ct where −△Ct = Ct (Target) – Ct (Reference). Fold change was presented by the 2−△△Ct method [13]. Sequences of all primers are listed on Additional file 1: Table S1.
Immunohistochemistry
Immunohistochemical staining for AZGP1 was performed in soft tissue sarcoma tissue microarray (TMA) by a standard two-step method. Briefly, the TMA sections were dried overnight at 37 °C, deparaffinized in xylene and rehydrated through a series of graded alcohol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 20 min. The slides were boiled in 10 mM sodium citrate buffer pH 6.0 by a pressure cooker for 10 min. After washing three times with phosphate buffered saline (PBS; 0.01 mol/L; pH = 7.4), the slides were incubated with 5% non-fat milk in PBS for 30 min to reduce nonspecific antibody binding. Subsequently, slides were incubated overnight at 4 °C with the rabbit polyclonal antibody against human AZGP1 (Abcam; Cambridge, UK; 1:100 dilution). After rinsing, the slides were incubated with goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) at a 1:100 dilution in PBS for 1 h at room temperature, and stained with 3,3-diaminobenzidine tetrahydrochloride (DAB). Finally, they were counterstained with Mayer’s hematoxylin, dehydrated in graded alcohols followed by xylene. Known immunostaining-positive specimens were used as positive controls and slides immunoreacted with PBS were used as the negative controls.
Western blotting
Total protein from cells was extracted in RIPA lysis buffer and quantified using BCA assay. 20 μg protein from each sample was separated by 10% SDS polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked in 5% non-fat milk for 1 h, washed three times with Tris-buffered saline containing 1% Tween 20 (TBST) at room temperature and then incubated overnight at 4 °C with the rabbit polyclonal antibody against human AZGP1 (Abcam; Cambridge, UK; 1:2000 dilution). After washing with TBST, membranes were incubated with secondary antibodies at room temperature for 1 h (goat anti-rabbit IgG, 1:10,000 dilution, Jackson ImmunoResearch Laboratories). Following washing with TBST, immunoreactivity was visualized by enhanced chemiluminescence reagents (Millipore). GAPDH served as internal reference.
Cell culture
Three human STS cell lines (RD ATCC® Number: HTB-166™, SW982 ATCC® Number: HTB-93™ and HT1080 ATCC® Number: CCL-121™) were purchased from American Type Culture Collection. RD and HT1080 cells were cultured in RPMI-1640 medium (Gibco, CA, USA) containing 10% fetal bovine serum (FBS; Gibco) at 37 °C with a humidified 5% CO2 atmosphere. SW982 cells were cultured in Leibovitz’s L-15 medium (Gibco, CA, USA) containing 10% FBS.
Vector construction
All constructs were made by standard DNA recombination techniques. The human AZGP1 (NM_001185.3) sequences were amplified by PCR from cDNA using primers listed in Additional file 1: Table S1, and subsequently cloned into lentiviral shuttle vector plenti6 (Invitrogen). For AZGP1 knockdown constructs, two short hairpin RNA (shRNA) sequences, including shRNA150 (target sequence: 5’-GGCTCACTCAATGACCTCCAG-3′), shRNA368 (target sequence: 5’-GTGAGATCGAGAATAACAGAA-3′) and scramble control sequence of 5’-GCTTCGCGCCGTAGTCTTA-3′ were designed and cloned into lentiviral shuttle plenti6-U6 vector.
Cell transfection
Lentiviral constructs were transfected into human HEK 293 T cells (ATCC® Number: CRL-11268™) with the ViraPower Packaging Mix (Invitrogen) to generate lentivirus. For infection, RD cells were seeded into 6-well plates at a density of 5 × 104 cells/well, and infected with AZGP1 over-expression lentivirus or empty lentivirus as control. HT1080 cells were infected with shRNA lentivirus or scramble lentivirus as control. Antibiotic-resistance cells were selected by 5 μg ml−1 blasticidin (Invitrogen) and used for subsequent experiment.
Wound healing assay
Cell spreading was analyzed using the wound healing assay. RD cell layers at 90% density in 24-well plates were scratched with a sterile 200 μL pipette tip and then washed with PBS. After 48 h, spreading cells were observed under the microphotography. Assays were repeated three times for each clone.
Transwell migration and invasion assay
Cell migration or invasion assay was performed in a 24-well Boyden chamber with or without Matrigel as described elsewhere [14]. The cells on the lower surface of the membrane were stained with crystal violet after fixation with 2% methanol for 5 min. Photographs of four randomly selected fields were taken to indicate cells that migrated to the other side of the membrane, and cell numbers were counted under a microscope at 200× magnification. Each test was performed in triplicate.
Statistical analysis
We calculated OS, 4-year, MFS and DSS using Kaplan-Meier analysis. We defined OS as the period between the date of the definitive surgery and the date of death, the 4-year survival as the period between the date of the definitive surgery and 4 years after, MFS as the interval between the date of the definitive surgery and the appearance of metastasis, and DSS as the date between the date of the definitive surgery and the time of death resulting from the disease itself. The effect of AZGP1 expression on Kaplan Meier survival curves was evaluated by the Log Rank test.
Mann Whitney and Kruskal Wallis tests were used to detect any association between AZGP1 mRNA expression and various pathological features (gender, age, TNM classification, recurrence, metastasis, and 4-year survival) between 2 or more groups, respectively.
Univariate analysis between pathological features (age, gender, AZGP1 expression, tumor size and histological grade) and metastasis or disease-specific survival was determined using Pearson’s correlation analysis. Multivariate analysis between the same variables was evaluated by the Cox regression model. Unpaired student’s T-test was performed to evaluate cell migration/invasion after gene modulation. All analyses were performed with SPSS® software 23.0 program for Windows® (SPSS Inc., Chicago, IL, USA). The statistical significance between groups was set at a p-value < 0.05.
